Project Summary/Abstract Mechanisms of PSGL-1 restriction of HIV virion infectivity Restriction factors are an important component of host innate immunity. Studying the anti-HIV mechanisms of restriction factors is central to understanding virus-host interaction. These mechanisms may also offer new therapeutic strategies to inactivate viral reservoirs to achieve lasting HIV remission. Recently, we have identified a new HIV restriction factor, PSGL-1 (P-selectin glycoprotein ligand-1), that can inactivate the infectivity of HIV virions released from HIV producing cells. PSGL-1 is a dimeric mucin-like 120-KD glycoprotein that is primarily expressed on the surface of lymphoid and myeloid cells. PSGL-1 binds to P-, L-, and E-selectin, and mediates leukocyte tethering and rolling on endothelium for leukocyte migration into inflamed tissues. PSGL-1 is also an INF-?-regulated factor involved in Th1-mediated anti-viral activity. Our preliminary mechanistic studies further revealed that PSGL-1 is incorporated into viral particles, which blocks HIV Env incorporation and disables the ability of virions to attach to target CD4 T cells for infection. In addition, we found that PSGL-1 is antagonized by Vpu and Nef through surface down-regulation. Based on these preliminary studies, we hypothesize that: (1) PSGL-1-mediated restriction of HIV infectivity involves its specific domains; (2) PSGL-1 restricts HIV infectivity likely through competitive exclusion of Env incorporation during viral assembly and steric hindrance of Env binding to cell receptors (3) Nef-mediated PSGL-1 down-regulation is likely achieved through linking PSGL-1 to components of clathrin-dependent trafficking pathways. In this application, we will pursue the following aims: Specific Aim 1 is to characterize PSGL-1 for inactivating HIV infectivity. We propose to identify PSGL-1 domains key to restricting HIV-1. We will determine the structure-function relationship of PSGL-1, defining the roles of PSGL-1 dimerization, N- terminal glycosylation and tyrosine sulfation, N-terminal decameric repeats, and the polybasic region in restricting HIV. Specific Aim 2 is to perform mechanistic studies of PSGL-1 inactivation of HIV viral infectivity. We hypothesized that PSGL-1 restricts HIV infectivity likely through two possible mechanisms: (1) competitive exclusion of Env incorporation during viral assembly; (2) steric hindrance of residual Env binding to cell receptors. We will test these two hypotheses to determine the mechanisms of action. Specific Aim 3 is to study the mechanism of Nef-mediated surface down-regulation of PSGL-1. We will identify functional domains of Nef and PSGL-1 for their involvement in PSGL-1 down-regulation. Nef-mediated PSGL-1 downregulation may facilitate viral spread in immune cells.